Traditional methods for the modification of gene expression in plants are characterized as “forward” and “reverse” genetic approaches. “Forward” genetic approaches include classical genetic analysis of naturally occurring or induced genetic variance in a plant or other organism. “Reverse” genetic approaches rely on the inactivation or modification of a specific gene.
Forward genetic approaches such as classical genetic techniques are limited by the available methods of detecting naturally occurring mutations and by methods of inducing mutations. Such techniques do not allow the researcher to target a specific gene for mutation but instead rely on the time-consuming process of screening large numbers of mutant plants or other organisms.
Available reverse genetic approaches require prior knowledge of the gene sequence. There are no reliable methods of gene replacement via homologous recombination available for use in higher plants. Methods of gene modification in plants instead rely upon a variety of alternative methods including insertional mutagenesis using “active” and “inactive” T-DNA species, as well as transposon mutagenesis. Additional methods include the production of sense or antisense transcripts using tissue-specific or constitutive regulatory elements.
The currently available techniques for reverse genetics in plants have many drawbacks. The methods are laborious and typically require generation and screening of large populations of transgenic plants. Further, these methods are not suitable for many recalcitrant plant species, including major crop species, where obtaining a large number of transgenic plants is difficult, impractical, or simply impossible. The art is thus in need of efficient, reliable methods for modifying gene expression in plants.